The present invention relates to autoinducer compounds which enhance gene expression in a wide variety of microorganisms. The present invention further relates to therapeutic compositions and therapeutic methods wherein, for example, gene expression within microorganisms is regulated.
Before 1981, microbiologists generally assumed that bacteria lacked the requirement and the capability of producing cell-cell signaling molecules. In 1981, by Eberhard, et al. Biochemistry, 20, 2444-2499, 1981, showed that the bacterium Photobacterium fischeri produces a compound 3-oxo-N-(tetrahydro-2-oxo-3-furanyl) hexanamide, also known as vibrio (photobacterium) autoinducer (VAI), which is associated with bacterial luminescence under conditions of high cell density. The cell membrane of P. fischeri was shown to be permeable to VAI by Kaplan and Greenberg in 1985 (J. Bacteriol., 163, 1210-1214, 1985). At low bacterial cell densities in broth medium, VAI passively diffuses out of the cells along a concentration gradient, where it accumulates in the surrounding medium. At high cell densities, the concentration of VAI outside the cells is equivalent to the concentration of VAI inside the cells. Under such conditions VAI was shown to initiate transcription of luminescence genes. Using such a system, bacteria are able to monitor their own population density and regulate the activity of specific genes at the population level.
For several years it was presumed that the autoinducer involved in bacterial luminescence was unique to the few bacteria that produce light in the marine environment. Then, in 1992, the terrestrial bacterium Erwinia carotovora was shown to use an autoinducer system to regulate the production of the xcex2-lactam antibiotic carbapenem (Bainton, et al., Biochem J., 288, 297-1004, 1992b). The molecule found to be responsible for autoinduction of carbapenem was shown to be an acylated homoserine lactone (HSL), a member of the same class of molecules responsible for autoinduction in bioluminescence. This finding led to a general search for HSLs in a wide range of bacteria To affect the search, a bioluminescence sensor system was developed and used to screen for HSL production in the spent supernatant liquids of a number of bacterial cultures. Many different organisms were shown by the screening to produce HSLs. These included: Pseudomonas aeruginosa, Serratia marcescens, Erwinia herbicola, Citrobacter freundii, Enterobacter agglomerans and Proteus mirabilis (Brainton, et al., Gene. 116, 87-91, 1992a; Swift, et al., Mol. Microbiol., 10, 511-520, 1993). More recently, the list has grown to include Erwinia stewartii (Beck, J. Bacteriol, 177, 5000-5008, 1993), Yersinia enterocolitica (Throup, et al., Mol. Microbiol., 17, 345-356, 1995), Agrobacterium tumefaciens (Zhang, et al., Nature, 362, 446-448, 1993), Chromobacterium violaceum (Winston, et al., Proc. Natl. Acad. Sci., USA, 92, 9427-9431, 1995), Rhizobium leguminosarium (Schripsema, et al., J. Bacteriol, 178, 366-371 1996) and others. Today it is generally assumed that all enteric bacteria, and the gram negative bacteria generally, are capable of cell density regulation using HSL autoinducers.
In 1993 Gambello, et al. Infect. Immun., 61, 1880-1184, (1993) showed that the xcex1-HSL product of the LasI gene of Pseudomonas aeruginosa controls the production of exotoxin A, and of other virulence factors, in a cell density dependent manner. Since that time, the production of a large number of Pseudomonas virulence factors have been shown to be controlled by xcex1-HSL compounds produced by the LasI and RhlI regulatory systems (Ochsner, et al., Proc. Natl. Acad. Sci., USA 92, 6424-6428, 1995; Winson, et al., supra; Latifi, et al., 1995), in a manner reminiscent of the Lux system. Latifi, et al. Mol. Microbiol, 21, 1173-1146, (1996) have also shown that many stationary phase properties of P. aeruginosa, including those controlled by the stationary phase sigma factor (RpoS), are under the hierarchical control of the LasI and RhlI cell-cell signaling systems.
In all cases, homoserine lactone autoinducers are known to bind to a DNA binding protein homologous to LuxR in Photobacterium fischeri, causing a conformational change in the protein initiating transcriptional activation This process couples the expression of specific genes to bacterial cell density (Latifi, et al. supra, 1996). Regulation of this type has been called xe2x80x98quorum sensingxe2x80x99 because it suggests the requirement for a xe2x80x98quoratexe2x80x99 population of bacterial cells before activation of the target genes (Fuqua, et al., J. Bacteriol., 176, 269-275, 1994b). Expression of certain of these xe2x80x98virulence factorsxe2x80x99 has been correlated with bacterial cell density (Finley and Falkow, Microbiol. Rev. 53, 210-230, 1989).
In P. aeruginosa, quorum sensing has been shown to be involved in the regulation of a large number of exoproducts including elastase, alkaline protease, LasA protease, hemolysin, cyanide, pyocyanin and rhamnolipid (Gambello, et al., supra; Latifi, et al., supra; Winson, et al., supra; Ochsner, et al., 1995). Most of these exoproducts are synthesized and exported maximally as P. aeruginosa enters stationary phase.
The concept of cell signaling and quorum sensing has been studied in the art. See for example U.S. Pat. No. 5,591,872, to Pearson et al.; Passador et al., Journal of Bacteriology, pages 5990-6000, October, 1996; PCT W092/18614 and U.S. Pat. No. 5,593,827.
Given the importance of these signaling molecules in the regulation of diverse metabolic functions, there exists a need for new autoinducer compounds which regulate gene expression in bacteria.
An object of the present invention is to provide novel autoinducer compounds and compositions comprising said novel compounds.
A further object of the invention is to provide novel methods for regulation, i.e., inhibition, enhancement, dispersion, etc., by administration of the compounds of the present invention.
Additional objects and advantages of the present invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from the practice of the invention The objects and advantages of the invention may be realized and obtained as particularly pointed out in the appended claims.
According to the present invention, the foregoing and other objects are achieved in part by compounds of the following formulae: 
wherein
R1-R21 are selected from the group consisting of H, C1-C4 alkyl group, OH, NH2, SH or a halogen, wherein the halogen is selected from the group consisting of fluorine, chlorine, bromine, and iodine;
R22 and R23 are selected from H, S, O, and Nxe2x80x94Y,
Y is selected from H, C1-C4 alkyl group, OH, NH2, SH or a halogen, wherein the halogen is selected from the group consisting of fluorine, chlorine, bromine, and iodine,
R24-R28 is H or a halogen, and
X, X1 and X2 are selected from O, S, NH, H2, H plus OH or NH2, or any combination of H plus one halogen or two halogens when one or more R groups is substituted, and at least one of R1-R21 is halogen, or the alkylene chain of the molecule contains a sulfur in the chain.
A further object of the present invention is to provide methods for regulating gene expression with a microorganism, which method comprises adding an inventive compound to a microorganism culture to cause expressing of a selected gene that would not otherwise be expressed.
Additional objects and advantages of the present invention will become readily apparent to those having ordinary skill in this art from the following detailed description, wherein only the preferred embodiment of the invention is shown and described, simply by way of illustration of the best mode contemplated for carrying out the present invention. As will be realized, the present invention is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
The present invention relates to autoinducer compounds of the formulae: 
wherein
R1-R21 are selected from the group consisting of H, C1-C4 alkyl group, OH, NH2, SH or a halogen, wherein the halogen is selected from the group consisting of fluorine, chlorine, bromine, and iodine;
R22 and R23 are selected from H, S, O, and Nxe2x80x94Y,
Y is selected from H, C1-C4 alkyl group, OH, NH2, SH or a halogen, wherein the halogen is selected from the group consisting of fluorine, chlorine, bromine, and iodine,
R24-R28 is H or a halogen, and
X, X1 and X2 are selected from O, S, NH, H2, H plus OH or NH2, or any combination of H plus one halogen or two halogens when one or more R groups is substituted, and at least one of R1-R21 is halogen, or the alkylene chain of the molecule contains a sulfur in the chain.
Included in the invention are optically active isomers of the claimed compounds as well as analogs of the claimed compounds. The term xe2x80x9cisomerxe2x80x9d includes molecules having the same molecular formula as the claimed compounds but possessing different chemical and physical properties due to a different arrangement of the atoms in the compound. Isomers include both optical isomers and structural isomers. The phrase xe2x80x9coptically activexe2x80x9d includes compounds that have the ability to rotate a plane of polarized light. An optically active isomer includes the L-isomer and the D-isomer of the claimed compounds.
The compounds of the present invention encompass compounds of formulae (I) and (II) modified as follows:
1) Alteration of the acyl side chain by increasing or decreasing its length.
2) Alteration of the structure of the acyl side chain, such as addition of a double bond or a triple bond between carbon atoms within the acyl side chain.
3) Substitution on carbons in the acyl side chain, e.g., the addition of a methyl group or other group such as an oxo-group, a hydroxyl group, an amino group, a sulfur atom, a halogen or dihalogen or some other atom or R-group to any location along the acyl side chain.
4) Substitution of carbons comprising the backbone of the acyl side chain with S or S substituted moieties or with N or N substituted moieties.
5) Substitution on the homoserine lactone ring portion of the molecule. For example: addition of a sulfur group to produce a thiolactone.
6) Halogenated acyl furanones have been shown to act as blockers to homoserine lactone cognate receptor proteins.
7) Ring size of the acyl side chain varying heterocyclic moiety is variable. For example, 4-membered and 6-membered rings containing nitrogen (i.e., beta and delta lactams) are included.
The following are specifically preferred compounds of the present invention: 
The present invention also relates to a method of regulating the expression of a gene. The method comprises inserting a gene into microorganisms chosen for enhancement of gene expression by an agent capable of stimulating the activity of a selected protein and incubating the microorganism with an agent capable of stimulating the activity of the selected protein. The method further can include the steps of allowing the gene expression to reach a desired level and then incubating the bacteria with an agent capable of inhibiting the activity of the selected protein.
Use is made of these compounds to control gene expression in microorganisms. The control exercised may be to decrease, inhibit, or increase, gene expression. The microorganisms concerned include bacteria, both Gram negative and Gram positive, yeasts and fungi, which have some gene whose expression is affected in some way by at least one of the inventive compounds.
Alternatively, a compound according to the present invention can be added to a microorganism culture in order to cause expression of a particular gene that would not otherwise be expressed. For example, the compound may be used to induce antibiotic production In yet another example, growth media for microorganisms can be prepared containing an autoinducer compound according to the present invention, at an effective concentration which would lead to a stimulation or promotion of the metabolism, growth and/or recovery of the organisms
A further method for utilizing the compounds disclosed in the present application is fully disclosed and described in copending U.S. application Ser. Nos. 09/591,476, filed Jun. 9, 2000, 09/319,580, filed Jul. 9, 1999.
The present invention further pertains to methods of inhibiting the infectivity of a selected microorganism, methods for treating an immunocompromised host infected by a microorganism, as well as therapeutic compositions. The methods comprise administrating to an individual a therapeutically effective amount of an agent that is capable of inhibiting the activity of a selected protein.
The language xe2x80x9cinhibiting the infectivity of a microorganismxe2x80x9d means methods of affecting the ability of the microorganism to initially infect or further infect an organism. This includes using agents that prevent a selected protein from activating the transcription of extracellular virulence factors.
The language xe2x80x9cagentxe2x80x9d means molecules that inhibit the ability of the selected protein to activate transcription of extracellular virulence factors. Inhibitory agents can be selected using method known to those having ordinary skill in the art.
The language xe2x80x9cadministering a therapeutically effective amountxe2x80x9d means methods of giving or applying an agent to an organism which allow the agent to perform its intended therapeutic function. The therapeutically effective amounts of the agent will vary according to factors such as the degree of infection in the individual, the age, sex, and weight of the individual, the ability of the agent to inhibit the activity of the selected protein of the microorganism in the individual. Dosage regimes can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. Administering also includes contacting the agent with the selected protein outside of an organism such as with a culture of bacteria.
The agent can be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the agent can be coated with a material to protect the agent from the action of enzymes, acids and other natural conditions which may inactivate the agent.
The agent can also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The pro-per fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the agent into a sterile vehicle which contains a basic dispersion medium and the required other ingredients enumerated above.
The agent can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The agent and other ingredients can also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the individual""s diet For oral therapeutic administration, the agent can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations may contain at least about 1% by weight of active component. The percentage of the compositions and preparations can, of course, be varied and can conveniently be between about 5 to about 80% of the weight of the unit. The amount of agent in such therapeutically useful compositions is such that a suitable dosage will be obtained.
The tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar or both. A syrup or elixir can contain the agent, sucrose as a sweetening agent, methyl and proplyparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the agent can be incorporated into sustained-release preparations and formulations.
The language xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d means solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the agent, use thereof in the therapeutic compositions and methods of treatment is contemplated. Supplementary active compounds can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of agent is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an agent for the treatment of microbial infection in individuals.
The principal agent is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
The language xe2x80x9can immunocompromised hostxe2x80x9d means an organism that has an immune system that has impaired capability of reacting to pathogens. The host can be immunocompromised due to a genetic disorder, disease or drugs that inhibit immune response.
The present invention is further illustrated by the following non-limiting examples. The contents of all of the references, published patent applications, and issued patents cited throughout this application are expressly incorporated by reference.